Summary: There is no successful biologic treatment for degenerative disc disease. The overall goal of this application is to establish an optimal cell-based solution to disc degeneration. This objective stems from the multiple failed attempts to regenerate discs using adult mesenchymal stem cells in clinical trials. Thus, we postulate that embryonic stem cell strategies are a novel approach to treating disc-related back pain. Our current knowledge indicates that cells from the notochord, which are of embryonic origin, initiate the differentiation of mesenchymal progenitors into disc forming cells during the development of the spine as well as in early childhood. However, notochordal cells cannot be derived from adult stem cells. Therefore, we believe that differentiation of notochordal cells from embryonic progenitors is necessary to successfully derive disc-forming cells. Specifically, we propose to derive cellular components of the inner compartments of the intervertebral disc (nucleus pulposus) from human embryonic stem cells (H9 and H1 lines) by generating mesenchymal-like stem cells and notochordal cells. Multiple ratios of these progenitors will be tested to form disc tissue components in vitro (Aim 1). The optimal ratio of hESC-derived notochordal and mesenchymal progenitor cells will be tested in a pre-clinical model of disc injury. Somatic cells will also be used as controls in these experiments (Aim 2). If successful, this high-risk, high-reward proposal bears a strong potential to develop new and effective cell/matrix-based therapeutic strategies for the regeneration of human intervertebral discs.